Mapping cytosine modifications

ABSTRACT

Methods, compositions and kits for selectively altering and detecting modified cytosine residues are provided.

CROSS REFERENCE

This application is a continuation-in-part of U.S. application Ser. No.13/826,395 filed Mar. 14, 2013.

GOVERNMENT RIGHTS

This invention was made with government support under GM096723 awardedby the National Institutes of Health. The government has certain rightsin this invention.

BACKGROUND

5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and5-carboxycytosine (5-caC) were recently identified in mammalian brainand embryonic stem cells as products of the oxidation of5-methylcytosine (5-mC) by cytosine oxygenases. The biological functionsof 5-hmC, 5-fC, and 5-caC are not completely understood; however,several lines of evidence suggest that 5-hmC is involved in epigeneticregulation and DNA demethylation. Iterative oxidation of 5-hmC bycytosine oxygenase enzymes yields 5-fC and 5-caC which are hypothesizedto be intermediates in the DNA demethylation process. Several challengesare associated with the identification these biologically modifiednucleobases in genomic DNA samples due to their low abundance andtemporal fluctuation. Mapping and quantifying 5-mC, 5-hmC, 5-fC, and5-caC at the DNA level is, therefore, important for unraveling theirrole in the dynamics of gene expression and regulation.

SUMMARY OF THE INVENTION

The present invention provides a variety of reagents, kits and methodsfor selectively altering and identifying modified nucleotides in anucleic acid such as DNA. The modified nucleotides that can beidentified include, for example, 5-mC, 5-hmC, 5-fC and 5-caC. Themethods, reagents and kits of the present invention permit not merelythe determination that a modified cytosine residue is present, but alsopermit the discrimination among or between different types of modifiednucleotides, such as 5-mC and 5-hmC, or between 5-hmC and 5-fC. In thisway, the invention can be used to elucidate the precise state of amodified nucleic acid, which may be a genome or genome fragment, forexample.

In some embodiments, the discrimination among or between differentoxidation states of a nucleotide is facilitated by alternatelyglucosylating or glucosaminylating various modified nucleotides. Theglucosylated or glucosaminylated forms can be distinguished based ontheir performance in various assays, such as by their differentialsensitivity to certain restriction endonucleases. In this way,alternately reacting the nucleic acid with a UDP-GlcN (Uridinediphosphate glucosamine or UDP-Glucosamine) substrate and a UDP-Glc(Uridine diphosphate glucose or UDP-Glucose) substrate, places off andon switches for endonuclease cleavage of a nucleic acid containingmodified nucleotides. By detecting the presence of cleavage-sensitivesites, the modified nucleotides can be located and identified.

In the present invention, the term glucosylation (and any form ofglucosylation such as “glucosylating” or “glucosylated”) refers to theincorporation of a glucosyl moiety from UDP-Glc into the 5-hydroxyposition of 5-hmC via the action of a glycosyltransferase to produce5-gmC (5-glucosyloxymethylcytosine). Other names in common use for 5-gmCinclude glucosyl-5-hydroxymethyl-cytosine,glucosyl-5-hydroxymethylcytosine, glucosyl-oxy-5-methylcytosine,5-glucosylhydroxymethylcytosine. In the present invention, the termglucosylation (and any form of glucosylation such as “glucosylating” or“glucosylated”) may also refer to the incorporation of an azido modifiedglucosyl moiety from UDP-Azido-Glc (for example, UDP-6-Azido-Glc orUDP-6-N3-Glc) into the 5-hydroxy position of 5-hmC via the action of aglycosyltransferase to produce a N3-5-gmC (for example,6-azide-glucosyl-5-hydroxymethylcytosine or 6-N3gmC). In the presentinvention, the term glucosaminylation (and any form of glucosaminylationsuch as “glucosaminylating” or “glucosaminylated”) refers to theincorporation of a glucosaminyl moiety from UDP-GlcN into the 5-hydroxyposition of 5-hmC via the action of a glycosyltransferase to produce5-gNmC (5-glucosaminyloxymethylcytosine). Other names for 5-gNmC includeglucosaminyl-5-hydroxymethyl-cytosine,glucosaminyl-5-hydroxymethylcytosine, 5-glucosylhydroxymethylcytosine,aminoglucosyl-5-hydroxymethyl-cytosine,aminoglucosyl-5-hydroxymethylcytosine, and5-aminoglucosylhydroxymethylcytosine. If the glycosyltransferase is aninverting glycosyltransferase such as T4 β-glucosyltransferase (BGT orβGT or beta-GT) the product is formed with a beta glycosydic linkage(for example, 5-β-gmC, 5-β-6-N3gmC, 5-β-6-gNmC, and 5-β-2-gNmC). If theglycosyltransferase is a retaining glycosyltransferase such as T4α-glucosyltransferase (AGT or aGT or alpha-GT) the product is formedwith an alpha glycosydic linkage (for example, 5-α-gmC).

Specifically, for example, the invention permits the differentiation of5-β-glucosyloxymethylcytosine (5-β-gmC) from5-β-2-glucosaminyloxymethylcytosine (5-β-2-gNmC) in a nucleic acid, byreacting the nucleic acid with an endonuclease capable of cleaving anucleic acid at a glucosylated nucleotide but not at an glucosaminylatednucleotide. One suitable endonuclease is AbaSI. Other usefulendonucleases include AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, andPxyI, or one of the ZZYZ proteins or its variants described in US PatentApplication Publication No. 2012/0301881, the complete disclosure ofwhich is hereby incorporated by reference. Accordingly, one of theseendonucleases, or a polypeptide at least 70% (e.g. at least 75%, atleast 80%, at least 82%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to one of thoseendonucleases or an active fragment thereof) can be used to selectivelycleave a target nucleic acid. By controlling the conversion ofglucosylation and glucosaminylation of various forms of naturallyoccurring modified nucleotides, the modified nucleotides can be locatedand identified in a manner distinguishing their original forms, such asmethylcytosine, hydroxymethylcytosine, formylcytosine, and/orcarboxycytosine.

Accordingly, in one aspect the invention provides methods forselectively altering modified nucleotides in a nucleic acid containinghydroxymethylated nucleotides and other modified nucleotides such as,5-fC or 5-caC. The methods generally include reacting a first subset ofthe modified nucleotides (e.g. 5-hmC) in the nucleic acid with UDP-GlcNin the presence of a β-glycosyltransferase, such asT4-β-glucosyltransferase, to convert hydroxymethylated nucleotides inthe nucleic acid to glucosaminylated nucleotides.

In some embodiments, the methods include the subsequent step of reactingthe nucleic acid with a reducing agent, such as NaBH₄, and a UDPderivative, such as UDP-Glc or UDP-azido-glucose, to convert a secondsubset of nucleotides in the nucleic acid to glucosylated orazido-glucosylated nucleotides. The reducing agent promotes thereduction of a nucleotide in a higher oxidation state, such as 5-fC or5-caC, to a hydroxymethylated nucleotide, which is then glucosylated orazido-glucosylated in a reaction typically catalyzed by aglycosyltransferase such as an α-glycosyltransferase or aβ-glycosyltransferase. The glycosyltransferase may or may not be thesame glycosyltransferase used to catalyze the previous conversion ofhydroxymethylated nucleotides to glucosaminylated nucleotides. Themethod can optionally differentiate pre-existing 5-hmC in a nucleic acidfrom newly formed as a result of the reduction of 5-fC or 5-caC, forexample. When a UDP-azido-glucose such as UDP-6-azido-glucose is used,5-hmC in the nucleic acid can be converted to aβ-6-azide-glucosyl-5-hydroxymethylcytosine in the presence of aβ-glycosyltransferase; Further derivatization of the azido moiety viaazide-alkyne Huisgen cycloaddition using a copper(I) catalyst (“clickchemistry”) or via copper-free variants (for example, using strainedcyclooctyne derivatives) can then optionally be used to label thenucleotide, such as with a biotin label, permitting the subsequent useof avidin to selectively isolate the labeled nucleotide.

In other embodiments, following the conversion of hydroxymethylatednucleotides in the nucleic acid to glucosaminylated nucleotides, thenucleic acid is reacted with an oxidizing agent, a UDP-Glc derivative,and a glycosyltransferase to convert a second subset of modifiednucleotides in the nucleic acid to glucosylated nucleotides. Suitableoxidizing agents include those capable of oxidizing 5-mC to 5-hmC, suchas mYOX1, a TET enzyme, or an inorganic oxidizing agent such as KRuO₄.The reaction with UDP-Glc is typically conducted in the presence of aglycosyltransferase such as an α-glycosyltransferase or aβ-glycosyltransferase. These methods can, for example, differentiatepre-existing 5-hmC in a nucleic acid from 5-hmC generated from a 5-mCprecursor.

In any of these methods, whether incorporating a reducing agent or anoxidizing agent, an endonuclease can be used to characterize thereaction products. Typically, the endonuclease is specific for theglucosylated (or azidoglucosylated) nucleotides, and not forglucosaminylated nucleotides, i.e., the endonuclease has a higherenzymatic activity for a glucosylated or azidoglucosylated nucleic acidthat it has for the same nucleic acid with an glucosaminylatednucleotide at the same position(s). Endonucleases that may be usedinclude, for example, AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI,and/or PxyI. An adapter molecule can then be ligated to the cleaved endof the endonuclease reaction product, facilitating the subsequentpurification, amplification or sequencing of the nucleic acid.

Accordingly, in one embodiment the invention provides a method fordifferentiating a 5-mC from 5-hmC in a genome or genome fragment. Thegenome or genome fragment may, for example, be mammalian in origin, suchas a human genome or genome fragment. The method includes reacting theisolated genome or genome fragment containing 5-mC and 5-hmC with (i)UDP-GlcN in the presence of a glycosyltransferase catalyzing transfer ofglucosamine to the 5-hmC; (ii) oxygenating any existing 5-mC residues to5-hmC by the action of TET or mYOX1; (iii) reacting the newly created5-hmC sites with UDP-Glc in the presence of a glycosyltransferasecatalyzing transfer of glucose to the 5-hmC; (iv) cleaving theglucosylated template with a modification-dependent endonuclease thatrecognizes at least one of the modified nucleotides; and (v)differentiating the original 5-mC from the 5-hmC by an altered cleavagepattern.

In this embodiment, the oxygenation of any existing 5-mC residues to5-hmC can be done in the presence of UDP-Glc and a glycosyltransferaseto catalyze the transfer of glucose to 5-hmC as it is being formed from5-mC. Alternatively, reaction conditions such as pH of the oxygenationreaction can be selected to optimize the yield of 5-hmC. See, forexample, Ser. No. 13/827,087, “Compositions and Methods for Oxygenationof Nucleic Acids Containing 5-Methylpyrimidine,” filed on the same dateas the present application and hereby incorporated by reference in itsentirety.

In another embodiment, the invention provides a method fordifferentiating a 5-mC from one or more of its oxidation products in agenome or genome fragment containing 5-mC and 5-hmC. The method includesreacting the isolated genome or genome fragment with UDP-2-glucosaminein the presence of a β-glycosyltransferase (BGT) catalyzing the transferof 2-glucosamine to the 5-hmC; reacting the isolated genome or genomefragment with mYOX1 or TET or a chemical oxidizing agent and optionallywith a reducing agent; cleaving the template with a modificationdependent endonuclease that is capable of selectively cleaving a 5-hmCand not a 5-glucosaminated hydroxymethylcytosine; and differentiatingthe 5-mC from one or more of its oxidation products by an alteredcleavage pattern.

The invention also provides preparations useful for convertingmethylcytosine or an oxidized nucleotide, such as 5-fC or 5-caC, or to aglucosylated nucleotide. The preparations include a reducing agent, suchas NaBH₄, or an oxidizing agent, such as mYOX1, a TET enzyme, or aninorganic oxidizing agent such as KRuO₄; a glycosyltransferase, such asan α-glycosyltransferase or a β-glycosyltransferase; a UDP-GlcN or a UDPderivative, such as UDP-Glc or UDP-azido-glucose.

The invention also provides preparations useful for modifying andselectively cleaving nucleic acids. The preparations include aglycosyltransferase and an endonuclease having an amino acid sequence atleast 95% identical to an enzyme such as AbaSI, AbaAI, AbaCI, AbaDI,AbaTI, AbaUI, AcaPI, and/or PxyI. The preparations also include (a)UDP-Glc and an oxidizing agent, or (b) UDP-GlcN. Where UDP-GlcN isincluded, the glycosyltransferase may be a BGT to catalyze the transferof glucose to hydroxymethylated pyrimidine residues. Where UDP-Glc andan oxidizing agent are included, the oxidizing agent, which may be amethylcytosine oxygenase such as mYOX1 or a TET enzyme or may be aninorganic oxidizing agent such as KRuO₄, promotes the conversion ofmethylcytosine residues in a nucleic acid to hydroxymethylcytosine,which can be glucosylated by the combination of UDP-Glc and theglycosyltransferase, and can be recognized by the endonuclease.

The invention also provides kits useful for making these preparationsand practicing these methods. For example, kits for modifyingformylcytosine or carboxycytosine residues in a nucleic acid can includea reducing agent, such as sodium borohydride, permitting the reductionof formylcytosine or carboxycytosine residues to hydroxymethylcytosine;a glycosyltransferase (such as a β-glycosyltransferase); and a UDPderivative, such as UDP-Glc and/or UDP-azidoglucose (such asUDP-6-azidoglucose), permitting the transfer of a sugar or modifiedsugar to the hydroxymethylcytosine. These kits may also include anoxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TETenzyme or an inorganic oxidizing agent such as KRuO₄) to promote theconversion of methylcytosine residues in a nucleic acid tohydroxymethylcytosine; UDP-GlcN; a restriction endonuclease (e.g.,having an amino acid sequence at least 95% identical to AbaSI, AbaAI,AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI); or any combination ofthe above.

Kits for selectively modulating the susceptibility of modified nucleicacid residues to cleavage can include UDP-Glc; UDP-GlcN; aβ-glycosyltransferase; and a reducing (e.g. sodium borohydride) oroxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TETenzyme or an inorganic oxidizing agent such as KRuO₄).

Kits useful for modifying and selectively cleaving nucleic acids caninclude, for example, a glycosyltransferase and an endonuclease havingan amino acid sequence at least 95% identical to an enzyme such asAbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI. These kitscan also include (a) UDP-Glc and an oxidizing agent, and/or (b)UDP-GlcN.

Some embodiments of the kits or preparations of the present inventioninclude an optional nucleic acid, such as a nucleic acid that is to bemodified, that is undergoing modification, and/or a control nucleicacid. Accordingly, a nucleic acid, if present, may include 5-hmC, 5-gmC,5-gNmC, 5-fC, 5-caC, 5-mC, or any combination of the above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that AbaSI recognizes 5-(β-glucosyloxymethyl)cytosine(5-β-gmC) but not 5-(β-2-glucosaminyloxymethyl)cytosine (5-β-2-gNmC)with high specificity as compared to 5-mC and C. The different forms ofcytosine modification (panels a-g) were digested by a 10-fold serialdilution of AbaSI enzyme (lane 1 to lane 7, lane 8 is undigestedcontrol).

FIG. 2 depicts the relative selectivity of a variety of endonucleasesfor nucleic acid substrates containing 5-β-gmC (“β-glucosylated hmC”),5-α-gmC (“α-glucosylated hmC”), 5-hydroxymethylcytosine (“hmC”),5-methylcytosine (“mC”), or unmodified cytosine (“C”).

FIG. 3 depicts selected cytosine modifications that can be achievedthrough the addition of glucose, modified glucose, or glucosamineresidues by T4-BGT alone or in combination withβ-glucosyl-α-glycosyltransferase from bacteriophage T6 (“T6-BGAGT”).

FIG. 4 depicts one example of an assay for detecting and/or mapping5-mC.

FIG. 5A-B depicts two examples of assays for detecting and/or mapping5-fC. The assay in FIG. 5A includes a step involving glucosylatinghydroxymethylcytosine residues newly-formed from the reduction of 5-fC.The assay in FIG. 5B includes a step involving azidoglucosylatinghydroxymethylcytosine residues newly-formed from the reduction of 5-fC.

FIG. 6 depicts an example of an assay for detecting and/or mapping5-caC.

FIG. 7 depicts LC-MS analyses of a nucleic acid containing 5-fC (toptrace) and the same nucleic acid after treatment with 100 mM sodiumborohydride (middle trace) or with sodium borohydride, UDP-Glc, andT4-BGT (bottom trace).

DETAILED DESCRIPTION OF EMBODIMENTS

Endonucleases have been identified from bacteria and more willundoubtedly be discovered using routine BLAST searches based on thepresent disclosure that are capable of preferentially cleaving 5-β-gmCcompared to C, 5-mC and 5-β-2-gNmC. In one example, the ZZYZ familymembers for example AbaSI (see for example WO 2011/091146),isoschizomers and mutants thereof preferentially cleave 5-β-gmC comparedto C, 5-mC and 5-β-2-gNmC. For example, AbaSI has cleavage activity at5-β-gmC that is 500 fold greater than 5-mC and 5-α-gmC. 5-β-gmC is theproduct of BGT mediated transfer of glucose from UDP-Glc to 5-hmC.

The specificity of AbaSI is demonstrated in the data shown in FIG. 1.AbaSI cleaves recognizes nucleic acids containing certain cytosinemodifications, cleaving them a short distance from those modifications.Ten-fold serial dilutions of AbaSI were combined with a PCR productcontaining cytosine (“C,” panel a), 5-methylcytosine (“5-mC,” panel B),5-hydroxymethylcytosine (“5-hmC,” panel C),5-(β-glucosyloxymethyl)cytosine (“5-β-gmC,” panel D),5-(β-2-glucosaminyloxymethyl)cytosine (“5-β-2-gNmC,” panel E),5-(β-6-glucosaminyloxymethyl)cytosine (“5-β-6-gNmC,” panel F), or5-(β-6-azidoglucosyloxymethyl)cytosine (“5-β-6-N3gmC,” panel G). The PCRproducts containing 5-β-gmC, 5-β-2-gNmC, 5-β-6-gNmC, or 5-β-6-N3gmC weregenerated by treatment of 5-hmC PCR DNA (panel c) withT4-β-glucosyltransferase (BGT) and the corresponding UDP-sugar (uridinediphospho-glucose (UDP-Glc), UDP-2-glucosamine (UDP-2-GlcN),UDP-6-glucosamine (UDP-6-GlcN), and UDP-6-N3-Glc, respectively). Theresults show that AbaSI does not digest C and 5-mC even at highconcentrations (panels a and b) whereas AbaSI can digest 5-β-gmC even atvery low concentrations (panel d). With respect to the threeglucosylated or glucosaminated cytosines, AbaSI can digest 5-β-6-gNmCand 5-β-6-N3gmC (panels f and g) more efficiently than it digests 5-hmC(although less efficiently than it digests 5-β-gmC. In contrast, it doesnot digest 5-β-2-gNmC (panel e).

Other endonucleases that differentiate between various forms of modifiedcytosine are also available, such as other ZZYZ family members describedin WO 2011/091146 and enzymes described in Borgaro et al. (2013)“Characterization of the 5-hydroxymethylcytosine-specific DNArestriction endonucleases,” Nucleic Acids Research, doi:10.1093/nar/gkt102, the entire disclosures of each of which areincorporated herein by reference. Several endonucleases thatdiscriminate among various cytosine modifications are described in FIG.2. For example, PvuRts1I cleaves nucleic acids containinghydroxymethylcytosine or glucosyloxymethylcytosine far more efficientlythan it cleaves nucleic acids containing only methylcytosine orunmodified cytosine. AbaSI, as described in the preceding paragraph,cleaves nucleic acids containing 5-β-gmC more efficiently than itcleaves nucleic acids containing 5-hmC, which are nevertheless cleavedmore efficiently than those nucleic acids containing only 5-mC orunmodified cytosine. Like AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI,AcaPI, and PxyI all demonstrate increased specificity for nucleic acidscontaining 5-β-gmC compared to nucleic acids containing only 5-hmC.Accordingly, for any of these enzymes β-glucosylation ofhydroxymethylcytosine can be used as an “on switch” to promote cleavagenear those positions, whereas β-glucosylation could be used as an “offswitch” for enzymes such as PvuRts1I, AbaBGI, BbiDI, BmeDI, or PatTI. Onthe other hand, α-glucosylation of 5-hmC generates a more efficientsubstrate for PvuRts1I, and could be used as an “on switch” to targetcleavage events to those locations in a nucleic acid.

It is expected that the use of endonucleases with preferentialspecificity for a specific modified nucleotide over other modified andunmodified nucleotides can be detected using the method described hereinfor 5-mC, 5-hmC, 5-fC, and 5-caC. In combination with the cofactorUDP-Glc, this system enables sequencing different epigenetic states of5-mC and greatly enhances the ability to determine the epigeneticmodification at a single base resolution level.

An embodiment of the method relies on modifying the non-target modifiedbase (for example, 5-hmC) chemically and/or enzymatically in such a waythat its reactivity to a endonuclease is completely or partiallyblocked, and then chemically or enzymatically reacting the targetmodified base to convert it into a newly formed 5-hmC (e.g., with areducing or oxidant agent) which can be then be reacted with aglycosyltransferase to form 5-gmC which in turn can be cleaved by anendonuclease that recognizes 5-gmC preferentially in a positiveidentification.

By the appropriate choice of the substrate for modifying the non-targetand target modified bases, this invention provides an on-off switchassay which is determined by enzyme specificity. For example, if in thefirst step UDP-2-GlcN is used to label pre-existing 5-hmC sites(resulting in an off-signal for AbaSI cleavage), and in the second stepthe sample is treated with a methylpyrimidine oxygenase (mYOX), BGT andUDP-Glc, an “on-signal” for AbaSI cleavage will be generated exclusivelyfor 5-mC sites which underwent oxidation by mYOX. If UDP-Glc is used tolabel pre-existing 5-hmC sites (on-signal for AbaSI cleavage), and thesample is treated with an mYOX, BGT and UDP-2-GlcN, then an “off-signal”will be generated for all 5-mC sites which were converted into 5-hmC bymYOX-mediated oxidation.

The 5-mC may be chemically or enzymatically converted to 5-hmC byreacting the 5-mC with mYOX1 (see for example, “nMCO1” described in U.S.Provisional Application No. 61/723,427), TET enzymes or chemicaloxidizing agents. Similarly, oxidation of 5-mC to 5-hmC to 5-fc to 5-CaCcan be achieved by chemical or enzymatic oxidation using mYOX1 or TET.Specific chemical oxidation of 5-hmC to 5-fC in synthetic nucleotideoligomer single strand (ssDNA) containing 5-hmC can be achieved withpotassium perruthenate, KRuO₄ (Booth, et al. Science, 336:934-937(2012)). KRuO₄ can oxidize 5-hmC in double-stranded DNA (dsDNA), with aninitial denaturing step before the addition of the oxidant, resulting inquantitative conversion of 5-hmC to 5-fC. Other oxidants known in art,such as Osmium (VIII)-based oxidants, Cerium (IV)-based oxidants, andChromium (VI)-based oxidants may be used for the oxidation of 5-hmC to5-fC.

A variety of sugars and modified sugars can be used to alter thepropensity of a 5-hmC to trigger an endonuclease-mediated cleavageevent. Some of these sugars and modified sugars are depicted in FIG. 3.For example, T4-BGT can be used to add glucose, 2-glucosamine,6-glucosamine, or 6-azidoglucose to 5-hmC. T4-BGT can also be used incombination with a β-glucosyl-α-glycosyltransferase, such as the onefrom bacteriophage T6, to generate disaccharidyl cytosine modificationsas shown in FIG. 3. By controlling the modifications made to aparticular nucleobase, the properties of that nucleobase can be changedin a manner facilitating its discrimination, whether through changes inits reactivity with an endonuclease; changes in the kinetics ofsynthesis of a complementary nucleic acid; or directly measured changesin size, shape, or charge density.

The reduction of 5-fC and its conversion to 5-β-gmC was demonstrated inthe experiment depicted in FIG. 7. Specifically, FIG. 7 is an LC-MSanalysis of a nucleic acid originally including 5-fC (top trace). In thepresence of sodium borohydride (NaBH₄), the 5fC is converted to 5-hmC(middle trace). When UDP-Glc and a BGT such as T4-BGT were alsoprovided, the 5-fC was converted all the way to 5-β-gmC, confirming thatthe modified forms of cytosine can be interconverted, facilitating theirsubsequent detection and discrimination.

In one embodiment the method comprises one or more of the followingsteps;

-   -   (a) Genomic DNA is treated with BGT and UDP-2-GlcN, so that all        5-hmC residues are converted to 5-β-2-gNmC.    -   (b) (i) The resulting DNA is treated with mYOX1, a Tet enzyme or        a chemical oxidant agent (converts 5-mC to 5-hmC), BGT and        UDP-Glc, so that all existing 5-mC residues are converted to        5-β-gmC; or        -   (ii) The resulting DNA is treated with a reducing agent            (e.g., NaBH₄, converts 5-fC to 5-hmC), BGT and UDP-Glc to            generate 5-β-gmC; or        -   (iii) The resulting DNA is treated with a reducing agent            (e.g., NaBH₄, converts 5-fC to 5-hmC), BGT and UDP-2-GlcN,            so that all 5-fC residues are converted to 5-β-2-gNmC. Then,            the resulting DNA is treated with a second reducing agent (a            different reducing agent or the same reducing agent but in            the presence of certain additives that converts 5-caC to            5-hmC), BGT, and UDP-Glc to generate 5-β-gmC.    -   (c) (i) The DNA is digested with a 5-β-gmC-dependent        endonuclease such as AbaSI, which cleaved at a fixed distance        from 5-β-gmC and left a sticky end (2-base 3′-overhang). Since        the endonuclease does not recognize C or 5-β-2-gNmC no cleavage        associated with these sites occurs. The only sticky ends created        are those resulting from 5-β-gmC residues, which in turn are        exclusively associated to 5-mC sites; or        -   (ii) The DNA is digested with endonuclease, which cleaves            5-β-gmC exclusively associated to 5-fCs sites, but not            5-β-2-gNmC, 5-mC, or C, leaving a sticky end (2-base            3′-overhang).        -   (iii) the DNA is digested with endonuclease, which cleaves            5-β-gmC exclusively associated to CaC sites, but not            5-β-2-gNmC, 5-mC, or C, leaving a sticky end (2-base            3′-overhang).    -   (d) A first adaptor (e.g., biotinylated adaptor A) is then        ligated onto the cleaved ends.    -   (e) The ligated DNA is then subjected to random fragmentation to        about 200 bp.    -   (f) Beads may be used to pull out the fragments with the ligated        adaptor. For example, avidin beads may be used to pull out the        biotin labeled adaptor (adaptor A). A person of ordinary skill        in the art will recognize other affinity systems and        immobilization matrices that can be used in place of        biotin/avidin beads.    -   (g) After polishing the ends, adaptor P is then ligated onto the        DNA fragments on the beads.    -   (h) The adaptor-specific PCR and the adapter ligated DNA enters        the library preparation pipeline for specific sequencing        platform where the end-sequencing is done from the adaptor A.

Reducing agents and conditions can be used herein that can convert thecarboxylic acids into alcohols, e.g., NaBH₄, CoCl₂, i-Pr₂NH, EtOH/H₂O(Jagdale, et al. Synthesis, 660-664 (2009)); EDC, HOBt, NaBH₄, THF/H₂O(Morales-Serna, et al. Synthesis, 1375-1382 (2011)); and cyanuricchloride, NaBH₄, NMM/H₂O (Falorni, et al. Tetrahedron Lett., 4395-4396(1999)) as well as other reducing agents known to a person of ordinaryskilled in the art.

Indeed, many water-soluble metal or metalloid hydrides are able toreduce aldehydes and/or carboxylic acids to alcohols. Examples of suchreducing agents are sodium borohydride and related compounds where from1 to 3 of the hydrogens are replaced by other moieties, such as cyanoand alkoxy containing up to about 5 carbon atoms. Examples ofsubstituted borohydrides, all of which are sodium, potassium, or lithiumsalts, include cyanoborohydride, dicyanoborohydride, methoxyborohydride,dimethoxyborohydride, trimethoxyborohydride, ethoxyborohydride,diethoxyborohydride, triethoxyborohydride, propoxyborohydride,dipropoxyborohydride, tripropoxyborohydride, butoxyborohydride,dibutoxyborohydride, tributoxyborohydride, and so forth. Examples ofother water-soluble metal hydrides include lithium borohydride,potassium borohydride, zinc borohydride, aluminum borohydride, zirconiumborohydride, beryllium borohydride, and sodiumbis(2-methoxyethoxy)aluminium hydride. Sodium borohydride can also beused in combination with a metal halide, such as cobalt(II), nickel(II),copper(II), zinc(II), cadmium (II), calcium (II), magnesium(II),aluminum(III), titanium (IV), hafnium(IV), or rhodium(III), each ofwhich can be provided as a chloride, bromide, iodide, or fluoride salt.Alternatively, sodium borohydride can be used in combination withiodine, bromine, boron trifluoride diethyl etherate, trifluoroaceticacid, catechol-trifluoroacetic acid, sulfuric acid, or diglyme.Particular reducing strategies include the combination of potassiumborohydride with lithium chloride, zinc chloride, magnesium chloride, orhafnium chloride; or the combination of lithium borohydride andchlorotrimethylsilane. Other reducing strategies include the use ofborane, borane dimethyl sulfide complex, borane tetrahydrofuran complex,borane-ammonia complex, borane morpholine complex, borane dimethylaminecomplex, borane trimethylamine complex, borane N,N-diisopropylethylaminecomplex, borane pyridine complex, 2-picoline borane complex, borane4-methylmorpholine complex, borane tert-butylamine complex, boranetriphenylphosphine complex, borane N,N-diethylaniline complex, boranedi(tert-butyl)phosphine complex, borane diphenylphosphine complex,borane ethylenediamine complex, or lithium ammonia borane. Alternativereducing strategies include the reduction of carboxylic acids via theformation of hydroxybenzotriazole esters, carboxy methyleniminiumchlorides, carbonates, O-acylisoureas, acyl fluorides, cyanurates, mixedanhydrides, arylboronic anhydrides, acyl imidazolide, acyl azides, orN-acyl benzotriazoles, followed by reaction with sodium borohydride togive the corresponding alcohols.

In one embodiment, 5-hmC sequencing can use BGT and UDP-Glc to generateAbaSI-active 5-β-gmC sites, and T4-α-glucosyltransferase (AGT) andUDP-Glc to generate AbaSI-inactive 5-α-gmC sites as a negative control.In another embodiment, 5-hmC sequencing can use BGT and UDP-Glc togenerate the AbaSI-active 5-β-gmC sites, and BGT and UDP-2-GlcN togenerate AbaSI-inactive 5-β-2-gNmC as a negative control for 5-hmC.

In one embodiment, newly generated 5-hmC sites can be differentiatedfrom pre-existing 5-hmC sites by sequentially transferring distinctsugar moieties from UDP-2-GlcN or native UDP-Glc using BGT.

In a further embodiment of the invention, UDP-Glc modified by any ofketo, thiol, chloro, fluoro, bromo, iodo, nitro, boron, and othersubstituents may be transferred onto 5-hmC using BGT and may block AbaSIactivity. Keto, thiol, chloro, fluoro, bromo, iodo, nitro, boron, andother substituents modifying glucose containing 5-hmC residues in anucleic acid may facilitate cytosine modification mapping and inhibitAbaSI cleavage.

All references cited herein are incorporated by reference.

EXAMPLES Example 1: Mapping of 5-hmC in a Nucleic Acid Sequence

The locations of 5-hmC in a nucleic acid sample can be determined usingdifferential cleavage.

A BGT can transfer glucose (“Glc”) from UDP-Glc to 5-hmC to form theglucoylated residue 5-β-2-gmC. Glucosylation enhances the sensitivity ofthe nucleic acid to a glucosylation-sensitive restriction enzyme such asAbaSI (see FIG. 2). Accordingly, the identification of 5-hmC sites in asample can be facilitating by glucosylating the nucleic acid, followedby identifying the locations of AbaSI cleavage sites.

Example 2: Exclusive 5-mC Methylome Mapping

An exemplary process for mapping 5-mC residues in a nucleic acid isdepicted in FIG. 4.

As shown in FIG. 4, genomic DNA is treated with a BGT andUDP-2-glucosamine, converting 5-hmC residues to 5-β-2-gNmC. Theresulting DNA is treated with a methylpyrimidine oxygenase fromNeisseria (mYOX1), TET or a chemical oxidizing agent, BGT and UDP-Glc,converting existing 5-mC residues to 5-β-gmC. The DNA is digested with a5-β-gmC-dependent restriction enzyme, such as AbaSI, cleaving at a fixeddistance from 5-β-gmC and leaving a sticky end (2-base 3′-overhang).Since AbaSI does not recognize C or 5-β-2-gNmC, the only sticky endscreated are those resulting from 5-β-gmC residues, which in turn areexclusively associated to 5-mC sites. A biotinylated adaptor A is thenligated onto the cleaved ends. The ligated DNA is then subjected torandom fragmentation to an average size of about 200 bp. Avidin beadsare used to pull out the fragments with the ligated adaptor A. Afterpolishing the ends, adaptor P is then ligated onto the DNA fragments onthe beads. The adaptor-specific PCR and the adapter ligated DNA entersthe library preparation pipeline for specific sequencing platform wherethe end-sequencing is done from the adaptor A.

Bioinformatic analysis of the sequencing reads is facilitated by thepresence of adapter A which marks the enzyme cleavage sites. Aftermapping the read back to the reference genome, the modified cytosine canbe mapped at fixed distance away from the cleavage sites.

Example 3: Exclusive 5-fC and/or 5-caC Mapping

Exemplary processes for identifying 5-fC or 5-caC residues in a nucleicacid are depicted in FIGS. 5A and 5B.

Example 3A: Glucosamination

As shown in FIG. 5A, a BGT can be used to catalyze the addition of2-glucosamine from UDP-2-GlcN to a 5-hmC residue, converting thoseresidues to 5-β-2-gNmC. A reducing agent such as NaBH₄ (optionally inthe presence of additives), can be used to reduce 5-fC and/or 5-caC to5-hmC. The newly formed 5-hmC when combined with BGT and UDP-Glc can besubsequently converted to 5-β-gmC.

As described in Example 2, the DNA is digested with a 5-β-gmC-dependentrestriction enzyme, such as AbaSI, cleaving at a fixed distance from5-β-gmC and leaving a sticky end (2-base 3′-overhang). Since AbaSI doesnot recognize C or 5-β-2-gNmC, the only sticky ends created are thoseresulting from 5-β-gmC residues, which in turn are exclusivelyassociated to 5-fC and/or 5-caC sites. A biotinylated adaptor A is thenligated onto the cleaved ends. The ligated DNA is then subjected torandom fragmentation to an average size of about 200 bp. Avidin beadsare used to pull out the fragments with the ligated adaptor A. Afterpolishing the ends, adaptor P is then ligated onto the DNA fragments onthe beads. The adaptor-specific PCR and the adapter ligated DNA entersthe library preparation pipeline for specific sequencing platform wherethe end-sequencing is done from the adaptor A.

Bioinformatic analysis of the sequencing reads is facilitated by thepresence of adapter A which marks the enzyme cleavage sites. Aftermapping the read back to the reference genome, the modified cytosine canbe mapped at fixed distance away from the cleavage sites.

Example 3B: 6-Azido-Glucose

Another exemplary process for mapping the locations of 5-fC and/or 5-caCis shown in FIG. 5B. The process depicted in FIG. 5B, like the processdepicted in FIG. 5A, begins with the addition of 2-glucosamine fromUDP-2-GlcN to a 5-hmC residue, converting those residues to 5-β-2-gNmCin a reaction catalyzed by a BGT, and the subsequent reduction of 5-fCand/or 5-caC residues to newly-generated 5-hmC residues. In the methoddepicted in FIG. 5B, UDP-6-azido-glucose (UDP-6-N3-Glc) is added tothose newly-generated 5-hmC residues by a BGT. The DNA is digested witha 5-β-gmC-dependent restriction enzyme, such as AbaSI, cleaving at afixed distance from 5-β-gmC and leaving a sticky end (2-base3′-overhang). Since AbaSI does not recognize C or 5-β-2-gNmC, the onlysticky ends created are those resulting from the azidoglucosylatedresidues, which in turn are exclusively associated to 5-fC and/or 5-caCsites.

The sticky ends are ligated to an adapter A. The resulting DNA isfragmented and ligated to a second adapter P1. The azido moiety canderivatized with a biotin label via azide-alkyne Huisgen cycloadditionusing a copper(I) catalyst (“click chemistry”) or via copper-freevariants (for example, using strained cyclooctyne derivatives) andavidin beads can then be used to selectively purify the fragmentscontaining the azidoglucosylated residues.

Example 4: Exclusive 5-caC Mapping

An exemplary process for mapping the locations of 5-caC in a nucleicacid sample is provided in FIG. 6.

As shown in FIG. 6, genomic DNA is treated with BGT and UDP-2-GlcN, sothat substantially all 5-hmC residues are converted to 5-β-2-gNmC. Theresulting DNA is treated with a reducing agent (e.g., NaBH₄, converts5-fC to 5-hmC), BGT and UDP-2-GlcN, so that all 5-fC residues areconverted to 5-β-2-gNmC. Then, the resulting DNA is treated with asecond reducing agent (a different reducing agent or the same reducingagent but in the presence of certain additives converts 5-caC to 5-hmC),BGT, and UDP-Glc to generate 5-β-gmC. The DNA is digested with arestriction endonuclease such as AbaSI, which cleaves 5-β-gmCexclusively associated to 5-caC sites but not 5-β-2-gNmC, 5-mC or C toleave a sticky end (2-base 3′-overhang). These sites are then identifiedthrough ligation to a biotinylated adapter, fragmentation andpurification using avidin-associated beads, ligation and analysis asdescribed in Example 2.

What is claimed is:
 1. A method for selectively altering any modifiedcytosine in a nucleic acid that is hydroxymethylcytosine (hmC) ormethylcytosine (mC), comprising: (a) reacting the nucleic acid withUDP-glucosamine in the presence of a glycosyltransferase to convert hmCin the nucleic acid to glucosaminylated cytosine, and (b) reacting thenucleic acid with an oxidizing agent and UDP-glucose to convert mC toglucosylated methylcytosine wherein the oxidizing agent is methylcytosine oxygenase or KRuO₄.
 2. The method according to claim 1, whereinthe reaction with UDP-glucose is catalyzed by a glycosyltransferase. 3.The method according to claim 1, further comprising cleaving the nucleicacid with an endonuclease specific for the glucosylated nucleotides andnot for glucosaminylated nucleotides.
 4. The method according to claim3, wherein the endonuclease has an amino acid sequence at least 95%identical to SEQ ID NO:
 3. 5. The method according to claim 3, whereinthe endonuclease has an amino acid sequence at least 95% identical to anenzyme selected from the group consisting of SEQ ID NOs: 3-8 and SEQ IDNO:
 10. 6. The method according to claim 3, comprising: ligating anadapter to the cleaved end of the nucleic acid.
 7. The method accordingto claim 1, wherein the methylcytosine oxygenase is a TET enzyme.
 8. Themethod according to claim 1, wherein the methylcytosine oxygenase ismYOX1.
 9. The method according to any one of claims 1-6, 7 and 8,comprising differentiating pre-existing 5-hydroxymethylcytosine fromnewly formed 5-hydroxymethylcytosine in a nucleic acid where5-methylcytosine is a precursor of 5-hydroxymethylcytosine.